The overall objectives of this proposal are to gain a better understanding of the molecular mechanism by which insulin regulates the changes in the activity of the enzyme tyrosine aminotransferase. Using a monospecific polyclonal antibody, a cDNA clone and a synthetic oligonucleotide complimentary to the first forty nucleotides of the mature transcript, the effect of insulin on protein stability, hybridizable mRNA, rates of transcription, and levels of functional mRNA will be investigated to attempt to reconcile the observations that insulin causes an increase in the enzyme activity but leads to a decrease in the amount of hybridizable mRNA. The mechanism by which insulin inhibits the glucocorticoid or cAMP mediated increase in the amount of hybridizable TAT mRNA will be investigated by measuring the stability of the TAT mRNA in the presence of insulin and the rates of transcription run-off in nuclei from hormone treated cells. Using a genomic clone and various subclones, the regions of the TAT gene capable of mediating the glucocorticoid induction of TAT will be identified. This will include not only the obvious 5' flanking sequence sites but also regions within the structural gene itself. Using these same clones studies will be undertaken to determine the interaction of insulin, glucocorticoids and cAMP in the regulation of TAT and to attempt to determine whether there are specific regions of the gene which might be insulin regulatory elements. Chimeric plasmids will be used to investigate the potential role of the multiple glucocorticoid regulatory elements or the putative cAMP regulatory elements as the regulatory element through which insulin acts in an antagonistic mode to glucocorticoids or cAMP or through which insulin might be mediating its own effects. This will involve using the plasmids as provided as well as modifying the plasmids to insert additional sequences as potential regulatory elements. These plasmids will be transfected into a cultured mammalian cell line already selected to require a drug resistant marker for survival under proper selection conditions.